RESUMO
Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
Assuntos
Mapeamento Cromossômico/métodos , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Genômica , Humanos , Locos de Características Quantitativas , Fatores de Transcrição/genéticaRESUMO
The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers. Hundreds of thousands of candidate enhancers have been nominated via biochemical annotations; however, only a handful of these have been validated and confidently linked to their target genes. Here we review emerging technologies for discovering, characterizing and validating human enhancers at scale. We furthermore propose a new framework for operationally defining enhancers that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements and CRISPR screens.
Assuntos
Elementos Facilitadores Genéticos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma Humano , HumanosRESUMO
Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.
Assuntos
Proteína BRCA1/genética , Edição de Genes , Predisposição Genética para Doença/classificação , Variação Genética/genética , Genoma Humano/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Linhagem Celular , Éxons/genética , Feminino , Genes Essenciais/genética , Humanos , Mutação com Perda de Função/genética , Modelos Moleculares , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reparo de DNA por Recombinação/genéticaRESUMO
Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to â¼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Lentivirus/genética , RNA Guia de Cinetoplastídeos/genética , Análise de Sequência de RNARESUMO
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.
Assuntos
Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica/genética , Hipoxantina Fosforribosiltransferase/genética , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência/genética , Linhagem Celular , Células HEK293 , Humanos , Hipoxantina Fosforribosiltransferase/biossíntese , Síndrome de Lesch-Nyhan/genética , RNA Guia de Cinetoplastídeos/genética , Tioguanina/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Distrofina/genética , Heme Oxigenase-1/biossíntese , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , GMP Cíclico/biossíntese , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Distrofina/deficiência , Heme Oxigenase-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , RNA Mensageiro/genética , Transdução de Sinais/genética , Citrato de Sildenafila , Sulfonas/farmacologia , Regulação para Cima , Peixe-Zebra/genéticaRESUMO
Chimeric genes can be caused by structural genomic rearrangements that fuse together portions of two different genes to create a novel gene. We hypothesize that brain-expressed chimeras may contribute to schizophrenia. Individuals with schizophrenia and control individuals were screened genome wide for copy-number variants (CNVs) that disrupted two genes on the same DNA strand. Candidate events were filtered for predicted brain expression and for frequency < 0.001 in an independent series of 20,000 controls. Four of 124 affected individuals and zero of 290 control individuals harbored such events (p = 0.002); a 47 kb duplication disrupted MATK and ZFR2, a 58 kb duplication disrupted PLEKHD1 and SLC39A9, a 121 kb duplication disrupted DNAJA2 and NETO2, and a 150 kb deletion disrupted MAP3K3 and DDX42. Each fusion produced a stable protein when exogenously expressed in cultured cells. We examined whether these chimeras differed from their parent genes in localization, regulation, or function. Subcellular localizations of DNAJA2-NETO2 and MAP3K3-DDX42 differed from their parent genes. On the basis of the expression profile of the MATK promoter, MATK-ZFR2 is likely to be far more highly expressed in the brain during development than the ZFR2 parent gene. MATK-ZFR2 includes a ZFR2-derived isoform that we demonstrate localizes preferentially to neuronal dendritic branch sites. These results suggest that the formation of chimeric genes is a mechanism by which CNVs contribute to schizophrenia and that, by interfering with parent gene function, chimeras may disrupt critical brain processes, including neurogenesis, neuronal differentiation, and dendritic arborization.
Assuntos
Variações do Número de Cópias de DNA , Genoma Humano , Proteínas Mutantes Quiméricas/genética , Esquizofrenia/genética , Adolescente , Adulto , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Estudos de Casos e Controles , Linhagem Celular , Criança , Deleção de Genes , Genes Duplicados , Predisposição Genética para Doença , Células HEK293 , Humanos , RNA Mensageiro/genética , Adulto JovemRESUMO
Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Transdução de Sinais , Receptores Proteína Tirosina Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Genômica , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/uso terapêuticoRESUMO
The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.
RESUMO
Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.
RESUMO
Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.
Assuntos
RNA Viral/genética , SARS-CoV-2/patogenicidade , Saliva/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.
RESUMO
Enhancers control the spatiotemporal expression of genes and are essential for encoding differentiation and development. Since their discovery more than three decades ago, researchers have largely studied enhancers removed from their genomic context. The recent adaptation of CRISPR/Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. In this Perspective, we discuss recent advances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools.
Assuntos
Sistemas CRISPR-Cas/genética , Elementos Facilitadores Genéticos/genética , Edição de Genes/métodos , Proteínas Associadas a CRISPR/metabolismo , Endonucleases/metabolismo , Edição de Genes/tendências , Humanos , Neisseria/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants.
Assuntos
Variação Genética , Genômica/métodos , Mutagênese , Proteínas/genética , Animais , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Moleculares , Fenótipo , Proteínas/química , Proteínas/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.